Blood coagulation factor VIII is a non-enzymatic glycoprotein cofactor that is essential for proper regulation of the blood coagulation cascade. Genetic deficiencies in factor VIII cause hemophilia A, which affects 1 in 5,000 males worldwide. Treatment for hemophilia A consists of repeated infusions of recombinant or plasma-derived factor VIII, which can result in the development of inhibitor antibodies that arise in approximately one-third of the hemophilia A patient population that receives therapy. The C2 domain of factor VIII is essential for proper interactions with von Willebrand factor and the membrane surface of activated platelets, and is a major epitope that is recognized by inhibitory antibodies. Recent studies indicate that a high incidence of the antibody response result in antibodies that block the activation of factor VIII by the serine proteases, thrombin or factor Xa. These "non-classical" antibodies possess a high level of pathogenicity and display positive cooperativity with "classical" antibodies, resulting in a dramatic decrease in the level of factor VIII activity. Much of the potency of the pathogenic response may correspond to the marginal stability of the human factor VIII protein. Indeed, factor VIII constructs that have increased stability, such as porcine factor VIII, can overcome the immune response and result in a modest level of factor VIII activity in circulation. This proposal aims to use structure-based computational protein design and x-ray crystallography to better understand the stability of the factor VIII C2 domain and the nature of its interactions with "nonclassical" antibody inhibitors. Additionally, we propose to understand the structural basis for cooperativity between the "classical" and "nonclassical" antibody types that target the factor VIII C2 domain. The specific aims of this project are: (1) Comparative study of the thermodynamic and functional properties of human and porcine FVIII C2 domains;(2) Structure-based computational protein design to engineer a thermo-stabilized FVIII C2 domain;(3) X-ray crystal structure of a human FVIII C2 domain/nonclassical antibody complex;and (4) X-ray crystal structure of a C2 domain/classical/nonclassical antibody ternary complex. PUBLIC HEALTH RELEVANCE: Hemophilia A is a bleeding disorder that affects 1 in 5,000 males worldwide. Upon treatment with factor VIII, inhibitor antibodies develop which often worsens their condition and makes therapy more difficult and expensive. This proposal aims to provide a better understanding of how these antibodies become pathogenic and will lead to the development of better treatments for hemophilia A patients. Hemophilia A is caused by the disruption of the gene encoding blood coagulation factor VIII and is the most common form of hemophilia, affecting 1 in 5,000 males worldwide. The most significant complication in the management of hemophilia A is the development of an inhibitory immune response to therapeutically infused factor VIII. Studying the structural basis of antibody recognition of factor VIII as well as using protein engineering to improve the stability of factor VIII may lead to improved therapies for hemophiliacs worldwide.